Inflammatory bowel diseases (IBD) are characterized by chronic intestinal inflammation. This persistent inflammation is the result of a poorly controlled mucosal immune response to normal intestinal microbiota. The mechanisms that initiate this aberrant response are not well elucidated; however, one possibility is that a change in the intestinal epithelial barrier results in increased systemic exposure to microbial products. The P-glycoprotein-deficient mouse is a unique model of colitis secondary to the altered function of a membrane hydrophobic pump. Our preliminary findings indicate that this P-glycoprotein-deficient model has an increase in colonic epithelial permeability, as well as a change in the sensitivity of toll-like receptors (TLRs) to bacterial products. We hypothesize that the loss of the intestinal epithelial pump, P-glycoprotein, alters the epithelial barrier and consequently the epithelial sensing of the microbiota by enhancing the ability of microbial products to interact with their specific TLRs. Our hypothesis includes the concept that in the normal intestine, expression of P-glycoprotein is maintained through the function of epithelial TLR9 and/or cyclooxygenase-2 (COX-2). TLR9 signaling induces COX-2 expression, COX-2 allows for PGE2 to dampen immune responses and upregulate P-glycoprotein expression, and P-glycoprotein functions to pump out toxic xenobiotics, maintain epithelial integrity, and maintain function of regulatory cells. In order to elucidate the relationships between epithelial membrane pumps, microbial sensors, and IBD we propose to: 1) determine the role of P-glycoprotein in the maintenance of intestinal epithelial barrier functions and in the composition and function of the mucosal immune system; 2) characterize the role of P-glycoprotein in the altered expression of TLRs and increased sensitivity to TLR-ligands seen in intestinal inflammation; and 3) investigate the contribution of TLR9 and COX-2 in the maintenance of intestinal epithelial barrier function and intestinal homeostasis. These studies will utilize both in vivo models of IBD, as well as in vitro primary and continuous epithelial cell culture systems. The understanding of the basic mechanisms by which the host maintains intestinal homeostasis and barrier integrity will lay the foundation for future studies on the regulation of the inflammatory response and the design of therapies for human IBD.